Enteroviruses, within the Picornaviridae family, are a genus of small, non-enveloped viruses containing positive-strand RNAs. The Enterovirus genus now comprises 12 species: Enterovirus A, Enterovirus B, Enterovirus C, Enterovirus D, Enterovirus E, Enterovirus F, Enterovirus G, Enterovirus H, Enterovirus J, Rhinovirus A, Rhinovirus B and Rhinovirus C. These viruses infect the intestinal tract but can cause various types of diseases. Typical enterovirus diseases are meningitis, paralysis, myocarditis, hand, foot and mouth-disease (HFMD), herpangina, pleurodynia, hepatitis, rash and respiratory diseases including pneumonia. The only enterovirus vaccine for use in human beings is vaccine of poliovirus which belongs to Enterovirus C. Currently, vaccines against non-polio enteroviruses are not available for human use.
Among Enterovirus A, enterovirus 71 (EV71) and coxsackievirus A group (CVA) infections are the most common causative factors of hand, foot, and mouth disease (HFMD) and other neurological disorders. Severe neurological disorders, including encephalitis, acute flaccid paralysis, pulmonary edema (PE), and hemorrhaging, culminating in fatality, particularly in EV71-infected children under 5 years old, have been reported [1-5]. Because EV71 and CVA infections can potentially become a new threat to global public health [1, 6-11], effective antiviral drugs and prophylactic vaccines are urgently needed.
Enterovirus genome consists of a single open reading frame that encodes the P1, P2, and P3 poly-proteins. The P2 and P3 regions encode nonstructural proteins (e.g., 3CD) responsible for virus replication and virulence. During viral RNA translation, the 2A protein catalyzes its N-terminal cleavage in cis, thereby releasing the capsid proteins in the P1 region from the nascent nonstructural proteins in the P2 and P3 regions. 3CD is released from the P3 precursor by autocatalytic cleavage. A 3C′ cleavage site in the polyprotein resides between the 3C and 3D portion of 3CD to generate 2 products, 3C′ and 3D′. When the P1 precursor is encoded by the P1 region, it can be cleaved by the 3C′ protease into VP0, VP1, and VP3. These 3 proteins spontaneously assemble into an icosahedral procapsid and pack the RNA genome into the provirion that could be a non-infectious empty (E)-particle or infectious full (F)-particle [12,13].
Human scavenger receptor class B, member 2 (hSCARB2) and human P-selectin glycoprotein ligand 1 (PSGL-1) have been identified as the important cell receptors for EV71 infection [14,15]. Our group [16] and Fujii et al. [17] have successfully developed transgenic mice expressing the human hSCARB2 receptor. In this promising model, transgenic animals infected with clinical EV71 isolates of the B4 and B5 subgenotypes developed HFMD-like skin rashes, whereas those inoculated with EV71 C2 and C4 subgenotypes or CVA16 suffered severe limb paralysis and death. In addition, passive administration of the monoclonal anti-EV71 VP1 neutralizing antibody N3 [26] reduced EV71 B5 infection-induced symptoms and protected the transgenic mice against EV71 C2-induced severe limb paralysis and death.
In a previous study [13], we produced a formalin-inactivated EV71 strain E59 (FI-EV71) vaccine candidate formulated with alum adjuvant, and found that FI-EV71 displayed high efficacy in the hSCARB2-Tg mouse challenge model [16]. In a human phase I clinical trial [18], FI-EV71 was safe and could elicit strong neutralizing antibody responses against current circulating EV71 isolates, but failed to protect against CVA16 infections. On the other hand, DNA vaccine (100 μg/mouse) and recombinant protein vaccine (10 μg/mouse) based on VP1, the most potent antigen on the EV71 virus, induce poorer immune responses than the inactivated virus vaccines and fail to effectively protect the mice against virus infection [19].
Virus-like particles, or VLPs, mimic the external protein structure of a virus without including the genetic material (DNA or RNA) that is necessary for viral replication. Without genetic material, VLP vaccines are incapable of causing infections themselves while at the same time presenting viral antigens in the most authentic configuration possible. VLPs of EV71 produced by insect cells also had been proved its efficacy in mice [20], which describes that VLP proteins are expressed in insect cells and then resemble to form VLPs and after purification, the VLPs are introduced to mice for immunization, inducing immune responses against the viral challenge. However, the different post-modification (such as glycosylation) of VLPs proteins produced from non-human cells might induce the different immunogenicity from human. The highest purity demand of VLPs during the production is also an obstacle.
There is still a need to develop an effective vaccine against enterovirus infections.